Physical exercise reverses immuno-cold tumor microenvironment via inhibiting SQLE in non-small cell lung cancer

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to February 2022. The therapeutic responses, evaluated according to the RECIST 1.1 criterion, were demarcated into four categories: complete response, partial response, stable disease, and progressive disease [7]. Ethical approval for the collection of samples was granted by the Clinical Research Ethics Committee at the Affiliated Wuxi People's Hospital of Nanjing Medical University (KY21126).

IHC and semi-quantitative evaluation
IHC staining was performed on the above TMAs and tissue slides. The primary antibodies used in this study were anti-SQLE (1:400, Cat. 12544-1-AP, ProteinTech, China) and anti-CD8 (1:200, Cat. GT2280, GeneTech, China). Antibody staining was visualized using diaminobenzidine and hematoxylin counterstain, and captured using Aperio Digital Pathology Slide Scanners [8]. The stained sections were independently evaluated by two pathologists using previously described criteria [9].

Cell culture and lentiviral transfection
The mouse Lewis lung carcinoma cell line was purchased from KeyGEN (Nanjing, China) and cultured at 37 °C with 5% CO2 in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. SQLE was overexpressed using lentiviral transfection according to the manufacturer's instructions [10]. Cells were seeded at 30% confluence in 6-well plates. After 24 h, cells in each well were transfected with virus solution (1 × 10 7 transduction units/ml) for 16 -24 h, incubated at 37 °C for 72 h, and assessed under a fluorescence microscope. Finally, 2 μg/ml puromycin was added to the complete medium to select stably transfected cell lines.

In vivo analysis
To investigate the effects of physical exercise on tumors, we first downloaded the GSE62628 dataset from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). It included gene expression profiles of melanoma tumor tissues from exercise and control groups of mice. The "limma" package in R was used to screen differentially expressed genes, and those with a false discovery rate < 0.05 were subjected to gene set enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery. The top 10 terms were displayed. The c2.cp.reactome.v7.4.symbols.gmt subclass from the Molecular Signatures Database [11] was used as the background.
The in vivo analysis was performed using 4 -6-week-old C57BL/6 mice (n = 30), which were purchased from Shanghai Laboratory Animal Co., Ltd. and commonly used for NSCLC immunotherapy research [12][13][14]. The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. All experiments were approved by the Laboratory Animal Ethics Committee at Nanjing Medical University (2022370). Lung cancer xenografts were established by subcutaneously injecting transfected homologous Lewis cells (1 × 10 7 ) into the flanks of mice. The injected mice were randomly assigned to either two groups (n = 5; Fig. 1c) or four groups (n = 5; Fig. 1m). Exercise intervention for mice included voluntary running on a wheel [15].
The tumors were regularly measured with calipers every 2 -3 d. On day 21, the mice were anesthetized with 0.5% sodium pentobarbital solution to remove the tumors [16], which were then photographed and weighed.
After euthanizing the mice, the tumors were carefully dissected and frozen in optimal cutting temperature compound, precooled using liquid nitrogen, and stored at -80°C until they were sectioned. Subsequently, 8-mm-thick serial cryosections were prepared for routine immunofluorescence staining of the target proteins [17,18] ab150160, Abcam, UK). Images were captured using a fluorescence microscope (ECHO Revolve, USA).

Mass cytometry
When mass spectrometry flow samples were selected, they were randomly numbered and the researcher conducting the on-board testing was unaware about their grouping. Mass cytometry was performed to analyze the samples obtained from three tumors per group, according to the procedure reported by Burnett et al. [19]. First, the cells were barcoded with distinct combinations of isotopically purified palladium ions that were chelated using isothiocyanobenzyl-EDTA. A total of 1 × 10 6 cells were treated with stable palladium isotopes in Maxpar Barcode Perm Buffer for 15 min on a shaker, washed twice using phosphate-buffered saline that contained 0.5% bovine serum albumin and 0.02% NaN3, and pooled into a single 15-ml tube.
The barcoded cells were treated with Fc Receptor Blocking Solution, stained at room temperature for 30 min with a surface antibody cocktail to highlight the target cells, washed twice with the same cell-staining media to remove any excess antibodies, permeabilized with methanol at 4 °C for 10 min, washed twice with the cell-staining media to remove methanol, incubated at room temperature for 1 h with the intracellular cocktail, washed twice with cell-staining media to remove any excess antibodies, treated overnight with 191/193Ir iridium intercalator solution diluted in phosphate-buffered saline with 4% Paraformaldehyde Fix Solution, and washed once with cell-staining media and twice with Cell Acquisition Solution before the mass cytometry run.
The samples were then diluted to approximately 1 × 10 6 cells/ml in Cell Acquisition Solution containing bead standards and analyzed using a Helios mass cytometer equilibrated with the same.
The cytometer collected approximately 0.5 × 10 6 cell events per sample at an even rate of 400 -500 events/s, with approximately 1.5 × 10 6 cell events in each group used for data analysis. The data were analyzed using Cytobank v8.1 (Beckman Coulter), and high-dimensional data were visualized using the t-distributed Stochastic Neighbor Embedding algorithm (5000 iterations, perplexity = 100) [20]. Table S1 provides a list of the antibodies and reagents used in this study.

Statistical analysis
Statistical analysis and figure exhibition were performed using R version 4.0.2 and Graphpad Prism 6.0. Continuous variables were analyzed using the Wilcoxon rank-sum test or the Mann-Whitney test, whereas categorical variables were assessed using the chi-squared test. Pearson's correlation was used to evaluate the correlation between two variables. The prognostic values of categorical variables were assessed using the log-rank test. For all analyses, a P-value < 0.05 was deemed to be statistically significant.  c Reactome analysis to identify pathways that the downregulated genes were enriched in. d Expression levels of genes in the "cholesterol biosynthesis" module (n = 5 per group). DEGs differentially expressed genes, RAB one protein from RAS oncogene family, GBP guanylate binding protein, GZMA granzyme A, GZMB granzyme B, HMGCR 3-hydroxy-3-methylglutaryl-coenzyme A reductase, SQLE squalene epoxidase